CD8+ T lymphocytes from healthy HIV-infected individuals show cytotoxic and noncytotoxic anti-HIV activities. Our laboratory has focused on the CD8+ cell noncytotoxic response (CNAR) that appears to be part of the innate immune system responding to HIV infection. When CD8+ cells from asymptomatic infected individuals are co-cultivated with HIV acutely infected CD4+ cells, suppression of virus replication takes place without killing the CD4+ cells. CNAR is not HLA-restricted, not specific for a particular retrovirus (inhibits all HIV-1, HIV-2 and SIV isolates tested), and appears very early in HIV infection. It is associated with secretion of an unidentified CD8+ cell antiviral factor (CAF), a protein stable to heat and low pH that inhibits HIV transcription. The clinical importance of CNAR/CAF activity has been shown in several studies of protection of individuals from HIV infection and disease progression. The specific objective of the present proposal is to identify the polypeptide(s) that mediates CAF activity. Our ultimate goal is to clone/sequence and produce CAF for evaluation in therapeutic and diagnostic trials. Proteomics and molecular studies are proposed. The proteomics approach involves mass spectrometric analysis of biochemically fractionated proteins from CAF-containing fluids. For these protein purification studies, we propose to use the chromatographic procedures we have developed thus far to separate CAF from many extraneous CD8+ cell culture proteins. Multidimensional chromatography-tandem mass spectrometric analysis and a stable isotype labeling of amino acids in culture (SILAC) will be employed to identify peptides unique or overexpressed in CAF-active fluids relative to control fluids. Standard protein purification procedures will also be conducted, where necessary, to further resolve CAF from other secreted CD8+ cell proteins. By the molecular approach, DMA microarray studies, confirmed by RT-PCR analyses, have identified 21 of the 107 candidate gene(s) found associated with CNAR/CAF activity. These genes are being further evaluated by transduction into 293T cells that are then assessed for production of anti-HIV proteins. In addition, candidate genes will be expressed in human GD8+ cells and the ability of these cells to suppress HIV replication and to produce CAF-like proteins will be tested. Finally, the clinical relevance of the identified protein(s) to CNAR/CAF will be established by shRNA studies and anti-sense mRNA alternative splicing procedures. The identification of CAF has outstanding potential as a therapeutic agent for HIV infection and for the development of an effective vaccine.